Effects of cell concentration during cryopreservation on the post-thaw quality of Santa ines ram sperm.

BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Ines rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G-400, G-800, G-1200, and G-1600 x 106 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5 degree C, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40 degree C per 20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G-400 treatment resulted in samples with the highest linearity and progressive motion (P < 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P < 0.05). Overall, use of the G-400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G-800 and G-1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single artificial insemination protocol is desirable. doi.org/10.54680/fr22610110812.

Origin and population structure of Brazilian hair sheep breeds.

Brazilian hair sheep constitute a genetic diversity hotspot. These animals are found in the harsh environments of the Brazilian Northwest (semi-arid) region. Genotypes (50K SNP chip) from seven Brazilian sheep breeds (five hair and two coarse wool types) and 87 worldwide breeds were used to test for population structure, admixture and genetic diversity. Moreover, phylogenetic trees evaluating migration events between genetic groups were built. Brazilian Somali, a fat-tailed breed, had a close relationship with East African breeds and clustered distinctly from other Brazilian breeds. Brazilian Blackbelly and Barbados Blackbelly had a close relationship. The Morada Nova breed did not show close relationships with European or African breeds, revealing a single migration event from an Algerian hair breed. Brazilian Fat-tail and Morada Nova share a common ancestor, but the former showed introgressions from Brazilian Somali and Afrikaner breeds, explaining the fat-tail phenotype. The Santa Inês breed received a substantial contribution from Brazilian Bergamasca and showed an admixed origin with recent introgressions from other breeds, mainly from Suffolk. Furthermore, Brazilian Somali and Brazilian Fat-tail are the most endangered sheep genetic resources in Brazil and should be the focus for ex situ conservation programs. In conclusion, Brazilian hair sheep show an African origin and are characterized by diverse genetic composition, reinforcing the need for conservation of these genetic resources, and at the same time, this highly diverse group has variability that can be used in breeding programs.

Effects of acute aerobic exercise on leukocyte inflammatory gene expression in systemic lupus erythematosus.

UNLABELLED: Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammation. Exercise induced inflammatory response in SLE remains to be fully elucidated. The aim of this study was to assess the effects of acuteexercise on leukocyte gene expression in active (SLEACTIVE) and inactive SLE (SLEINACTIVE) patients and healthy controls(HC). METHODS: All subjects (n = 4 per group) performed a 30-min single bout of acute aerobic exercise (~70% of VO2peak) on a treadmill, and blood samples were collected for RNA extraction from circulating leukocyte at baseline, at the end of exercise, and after three hours of recovery. The expression of a panel of immune-related genes was evaluated by a quantitative PCR array assay. Moreover, network-based analyses were performed to interpret transcriptional changes occurring after the exercise challenge. RESULTS: In all groups, a single bout of acute exercise led to the down-regulation of the gene expression of innate and adaptive immunity at the end of exercise (e.g., TLR3, IFNG, GATA3, FOXP3, STAT4) with a subsequent up-regulation occurring upon recovery. Exercise regulated the expression of inflammatory genes in the blood leukocytes of the SLE patients and HC, although the SLE groups exhibited fewer modulated genes and less densely connected networks (number of nodes: 29, 40 and 58; number of edges: 29, 60 and 195; network density: 0.07, 0.08 and 0.12, for SLEACTIVE, SLEINACTIVE and HC, respectively). CONCLUSION: The leukocytes from the SLE patients, irrespective of disease activity, showed a down-regulated inflammatory geneexpression immediately after acute aerobic exercise, followed by an up-regulation at recovery. Furthermore, less organized gene networks were observed in the SLE patients, suggesting that they may be deficient in triggering a normal exercised-induced immune transcriptional response.

Genetic diversity of the forage peanut in the Jequitinhonha, São Francisco, and Paranã River valleys of Brazil.

Arachis pintoi and A. repens are legumes with a high forage value that are used to feed ruminants in consortium systems. Not only do they increase the persistence and quality of pastures, they are also used for ornamental and green cover. The objective of this study was to analyze microsatellite markers in order to access the genetic diversity of 65 forage peanut germplasm accessions in the section Caulorrhizae of the genus Arachis in the Jequitinhonha, São Francisco and Paranã River valleys of Brazil. Fifty-seven accessions of A. pintoi and eight of A. repens were analyzed using 17 microsatellites, and the observed heterozygosity (HO), expected heterozygosity (HE), number of alleles per locus, discriminatory power, and polymorphism information content were all estimated. Ten loci (58.8%) were polymorphic, and 125 alleles were found in total. The HE ranged from 0.30 to 0.94, and HO values ranged from 0.03 to 0.88. By using Bayesian analysis, the accessions were genetically differentiated into three gene pools. Neither the unweighted pair group method with arithmetic mean nor a neighbor-joining analysis clustered samples into species, origin, or collection area. These results reveal a very weak genetic structure that does not form defined clusters, and that there is a high degree of similarity between the two species.

Genetic diversity and assessment of 23 microsatellite markers for parentage testing of Santa Inês hair sheep in Brazil.

Santa Inês is the most common hair sheep breed in Brazil and probably has the highest genetic diversity among sheep breeds in this country. Successful breeding programs for Brazilian sheep breeds are not common for various reasons, including a lack of control of parentage in the flocks. We developed an allele frequency database for 23 STR loci for the Santa Inês breed based on 285 animals sampled from five populations distributed across the central-western and north-eastern regions of Brazil. The marker set included seven microsatellites used in the 2011 International Society for Animal Genetics sheep genotyping comparison tests and all eight microsatellites currently approved by the Brazilian Agricultural Ministry laboratory accreditation guidelines for sheep identification. The microsatellites had an average of 10 alleles and a mean expected heterozygosity of 0.745. Combined paternity exclusion probabilities when no parent or one parent was known were >99.99%. A small proportion (5.8%) of the existing genetic variation was found to be among the Santa Inês populations, possibly derived from genetic drift and selection. We found that the marker panel proposed by the Agricultural Ministry, although generally useful, should be enhanced by including more markers for improved exclusionary power in parentage testing. This database provides a useful tool for parentage testing of this major Brazilian breed, contributing to improved management and breeding of existing herds.

Cryopreservation of tambaqui (Colossoma macropomum) semen: extenders, cryoprotectants, dilution ratios and freezing methods.

The tambaqui is an Amazonian fish of great economic and environmental importance to Brazil and other South American countries. Several semen cryopreservation methodologies have been tested for different Brazilian fish species; however, there is little information on the use of this technique on tambaqui semen. The aim of the present study was to investigate the effect of osmolarity and activation solutions on sperm kinetics and, glucose solutions, cryoprotectants, dilution ratios, egg yolk and freezing methods on tambaqui semen freezing. The osmolarity of 230 mOsm was suitable for simultaneously yielding higher sperm motility (85%) and motility time (54 sec.) and osmolarities above 360 mOsm maintain immobile tambaqui sperm. The tambaqui semen can be successfully cryopreserved when diluted 1:9 in freezing medium composed of 5 percent glucose solution (290 mOsm) with 10 percent methylglycol and 5 percent egg yolk, and frozen directly in a dry shipper container.

A new polymorphism in the Growth and Differentiation Factor 9 (GDF9) gene is associated with increased ovulation rate and prolificacy in homozygous sheep.

Brazilian Santa Inês (SI) sheep are very well-adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are the first two genes expressed by the oocyte to be associated with an increased ovulation rate in sheep. All GDF9 and BMP15 variants characterized, until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study, we have found a new allele of GDF9, named FecG(E) (Embrapa), which leads to a substitution of a phenylalanine with a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecG(E) allele have shown an increase in their ovulation rate (82%) and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.

Genotoxicity of SPL (spent pot lining) as measured by Tradescantia bioassays.

Spent Pot Liner (SPL) is a solid waste product generated in the process of aluminum production. Tradescantia micronuclei (Trad-MN) and stamen hair mutation (Trad-SHM) bioassays are very useful tests to assess genotoxicity of environmental pollutants. In the present study, we intended to investigate the genotoxicity of this waste with Tradescantia bioassays using leachates of SPL simulating the natural leachability of SPL in soil. The formation of micronuclei (MN) was found to be concentration dependent. MN frequency enhanced significantly with SPL treatment. In addition, SPL also appeared to increase the percentage of dyads and triads. Trad-SHM assay showed that SPL increases pink mutation events as SPL concentration increases. These results demonstrated that SPL is a cytogenotoxic agent that affects different genetic end-points (induction of micronuclei and point mutations) even at low concentration (2% and 3%).

Starch-poly-epsilon-caprolactone microparticles reduce the needed amount of BMP-2.

BMP-2 is currently administered clinically using collagen matrices often requiring large amounts of BMP-2 due to burst release over a short period of time. We developed and tested a novel injectable drug delivery system consisting of starch-poly-epsilon-caprolactone microparticles for inducing osteogenesis and requiring smaller amounts of BMP-2. We evaluated BMP-2 encapsulation efficiency and the in vitro release profile by enzyme-linked immunosorbent assay. BMP-2 was rapidly released during the first 12 hours, followed by sustained release for up to 10 days. We then evaluated the osteogenic potential of dexamethasone (standard osteogenic induction agent) and BMP-2 after incorporation and during release using an osteo/myoblast cell line (C2C12). Alkaline phosphatase activity was increased by released BMP-2. Mineralization occurred after stimulation with BMP-2-loaded microparticles. A luciferase assay for osteocalcin promoter activity showed high levels of activity upon treatment with BMP-2-loaded microparticles. In contrast, no osteogenesis occurred in C2C12 cells using dexamethasone-loaded microparticles. However, human adipose stem cells exposed to the microparticles produced high amounts of alkaline phosphatase. The data suggest starch-poly-epsilon-caprolactone microparticles are suitable carriers for the incorporation and controlled release of glucocorticoids and growth factors. Specifically, they reduce the amount of BMP-2 needed and allow more sustained osteogenic effects.

Single nucleotide polymorphisms in the growth hormone and IGF type-1 (IGF1) genes associated with carcass traits in Santa Ines sheep.

Polymorphisms in the growth hormone (GH) and IGF type-1 (IGF1) genes have been associated with the economic traits in farm animals, including BW of some sheep breeds. However, it remains unknown if these polymorphisms also affect carcass traits in sheep. Thus, we aimed to identify polymorphisms in the GH and IGF1 genes in Santa Ines sheep in order to describe their allelic and genotypic frequencies as well as to test the hypotheses that they are associated with the carcass traits. Fragments of 4550 bp (IGF1) and 1194 bp (GH) were sequenced in up to 191 lambs. In all, 18 polymorphisms were identified in the IGF1 and 21 in the GH gene. The IGF1 polymorphisms rs430457475, rs412470350, rs409110739 and rs400113576 showed an additive effect on the internal carcass length (-0.9265±0.4223), rump girth (-2.9285±1.1473), rib yield (-1.0003±0.4588) and neck weight (-0.0567±0.0278), respectively. In addition, the polymorphisms rs58957314 in the GH affected the rib weight (-0.4380±0.1272) and rib yield (-2.2680±0.6970), loin weight (-0.1893±0.0516) and loin yield (-0.9423±0.3259), palette weight (-0.2265±0.0779) and palette yield (-0.9424±0.4184), leg weight (-0.3960±0.1375), neck weight (-0.0851±0.0394) and carcass finishing score (-0.1700±0.0839). These results allow us to conclude that there are polymorphisms in the IGF1 and GH genes associated with carcass traits in Santa Ines sheep, which can provide important information for marker-assisted selection.

Evaluation of morphology, morphometry and follicular dynamics in FecGE genotyped ewes.

This study aimed to evaluate the effect of FecGE mutation on the development of ovarian follicles. To this end, 42 Santa Inês ewes were genotyped for FecGE mutation and classified as wild-type (FecG+/+), heterozygous (FecG+/E) or mutant homozygous (FecGE/E). Ovarian fragments were processed, and the follicles were analyzed with regard to the morphology and morphometry using classical histology. For the evaluation of follicular dynamics, ewes underwent oestrous synchronization and were monitored throughout an interovulatory period. A higher (P < 0.05) percentage of morphologically normal follicles in the primordial stage was identified in FecGE/E (90.0%) and FecG+/E (88.1%) ewes than in the FecG+/+ (73.0%) ewes. There was also a significantly greater (P < 0.05) number of morphologically normal follicles in the FecGE/E (87.3%) and FecG+/E (83.3%) ewes than in FecG+/+ (76.8%) ewes in the transitional stage. A smaller (P < 0.05) diameter was observed in the secondary follicles in FecGE/E (93.8 µm) ewes than in FecG+/E (171.8 µm) ewes. Regarding follicular dynamics, FecGE/E ewes showed a greater (P < 0.05) number of ovulations (2.5 ±â€¯0.2) than FecG+/+ ewes (1.5 ±â€¯0.3) ewes. Ovulatory follicles were smaller (P < 0.05) in the FecGE/E (5.1 mm) and FecG+/E (5.2 mm) ewes than in FecG+/+ (5.8 mm) ewes. Santa Inês nulliparous ewes carrying the FecGE mutation showed a greater proportion of morphologically normal follicles in the primordial and transitional stages than those not carrying the mutation. FecGE/E ewes demonstrated a higher number of ovulated follicles and that FecGE/E and FecG+/E ewes presented ovulatory follicles with a smaller diameter.

Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends.

The fields of tissue engineering and regenerative medicine aim at promoting the regeneration of tissues or replacing failing or malfunctioning organs, by means of combining a scaffold/support material, adequate cells and bioactive molecules. Different materials have been proposed to be used as both three-dimensional porous scaffolds and hydrogel matrices for distinct tissue engineering strategies. Among them, polymers of natural origin are one of the most attractive options, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as typically good biological performance. In this review, the most studied and promising and recently proposed naturally derived polymers that have been suggested for tissue engineering applications are described. Different classes of such type of polymers and their blends with synthetic polymers are analysed, with special focus on polysaccharides and proteins, the systems that are more inspired by the ECM. The adaptation of conventional methods or non-conventional processing techniques for processing scaffolds from natural origin based polymers is reviewed. The use of particles, membranes and injectable systems from such kind of materials is also overviewed, especially what concerns the present status of the research that should lead towards their final application. Finally, the biological performance of tissue engineering constructs based on natural-based polymers is discussed, using several examples for different clinically relevant applications.

Fish sperm subpopulations: Changes after cryopreservation process and relationship with fertilization success in tambaqui (Colossoma macropomum).

Fish tambaqui (Colossoma macropomum) is the native Brazilian fish with the highest agricultural production under intensive aquaculture in South America. However, the decrease in the genetic variability in fish farms has become necessary the improvement of cryopreservation process through new statistical studies of spermatozoa (like subpopulation studies). The evaluation of the kinetic data obtained with a computer-assisted sperm analysis system, applying a two-step cluster analysis, yielded in tambaqui three different subpopulations in fresh sperm: SP1, considered as a slow nonlinear subpopulation; SP2, considered as a fast nonlinear subpopulation, and finally; SP3, considered as a fast linear subpopulation. For cryopreserved sperm, the cluster analysis yielded only two sperm subpopulations: SP1', considered as a slow nonlinear subpopulation and SP2', which seemed to be an intermediate subpopulation (showing medium motility and velocity values) merged from SP2 and SP3 obtained from fresh sperm. Coefficients of correlation (r) and determination (r2) between the sperm subpopulations from fresh sperm and the fertilization rates were calculated, and SP2 and SP3 (the fast-spermatozoa subpopulations) showed a high-positive correlation with the fertilization rates (r = 0.93 and 0.79, respectively). In addition, the positive significant correlations found in curvilinear velocity (r = 0.78), straight line velocity (r = 0.57), and average velocity (r = 0.75) indicate that sperm kinetic features seem to be a key factor in the fertilization process in tambaqui, as occur in other fish species.

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFß1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage. Copyright © 2015 John Wiley & Sons, Ltd.

A new defined medium for cultivating Leishmania promastigotes.

A chemically defined medium to grow the promastigotes of 19 stocks of Leishmania is described. The medium was developed by making qualitative and quantitative modifications of the medium AR-103 devised for Trypanosoma cruzi. The new medium, designated as MD-29, can be used in studies on the nutritional requirements of promastigotes.

Evaluation of the 226Ra transport by river sediments surrounding the Brazilian uranium mining and milling facilities.

A study of the (226)Ra contamination of the river sediments surrounding the Brazilian uranium mining and milling facilities was carried out. The nondetrital (226)Ra concentrations were determined performing a 0.5 N HCl leaching. Some preliminary speciations experiments using Tessier's sequential extraction procedure were also carried out. (228)Ra was used as a monitor of the natural variations. In general one could not observe increase in the non-detrital (226)Ra fraction between the pre-operational and operational data. Additionally, speciations experiments have shown the exchangeable fraction to be the main one responsible for (226)Ra content. Although the results indicate that the sediment is an important means of radium downstream transport, its availability suggests the soluble form to be the main path through the main foodchain. Attention must be paid to the release of (226)Ra bound to barium sulphate from the chemical processing effluent since increments of that fraction were observed in bottom sediments.

Pre-operational environmental survey at the uranium mine and mill site, Poços de Caldas, Minas Gerais, Brazil.

The first Brazilian uranium mine and mill are located on the Pocos de Caldas plateau, in the Central State of Minas Gerais. The pre-operational environmental survey was carried out over a period of two years by the Brazilian Nuclear Energy Commission (CNEN), through the Instituto de Radioprotecão e Dosimetria (IRD). The selection of the materials to be monitored, the sampling points and the radionuclides to be analysed were based on critical parameters taking into account the meteorological and hydrological characteristics of the site as well as land use. The results obtained are sufficient to characterize the environmental background of the area, and will allow an assessment of the environmental impact due to the operation of the facility and an evaluation of the adequacy of the effluent emission control measures. In relation to their contribution to the population dose, the results indicated that 226Ra and 210Pb will be the important radionuclides.

Effects of ibopamine eye drops on intraocular pressure and aqueous humor flow in healthy volunteers and patients with open-angle glaucoma.

PURPOSE: On the basis of intraocular pressure measurements and fluorophotometry we assessed the effects of 2% ibopamine eye drops on aqueous humor production in normal and glaucomatous eyes. METHODS: Thirty subjects (15 healthy volunteers and 15 open-angle glaucoma patients with ocular hypertension) were included in a placebo-controlled study with random assignment of treatment from masked containers. All subjects underwent ophthalmologic examinations and intraocular pressure (IOP) measurements. Fluorophotometry was done in both eyes at baseline (without treatment) and during treatment. Each subject was treated with 1 drop of 2% ibopamine in one eye and 1 drop of placebo in the fellow eye 30 minutes before fluorophotometric scans and every hour after the first instillation (for a total of 4 times). Safety was evaluated by recording adverse events and ocular symptoms and signs. Aqueous humor flow data were analyzed using the paired t-test, comparing ibopamine and placebo-treated eyes. RESULTS: No changes in IOP were detected in normal eyes, whereas glaucomatous eyes showed a mean increase of 4 mmHg (95% CI 3.46-4.51) from baseline. The difference in IOP between healthy eyes and those with glaucoma was significant (p < 0.0001). In normal eyes and patients with glaucoma ibopamine led to a significant increase in aqueous humor flow compared with placebo-treated eyes (p < 0.01). The safety profile of ibopamine was very good. CONCLUSIONS: The results seem to confirm that ibopamine increases aqueous humor production in normal and glaucomatous eyes, raising IOP only in eyes with glaucoma.

[The treatment of cryptosporidiosis in AIDS patients using paromomycin]. / Tratamento da criptosporidíase em pacientes com AIDS, por meio da paromomicina.

The authors treated with paromomycin 25 patients, with AIDS and cryptosporidiosis. The drug was given orally in a doses of 500 mg qid, for a period of 14 days. Tolerance was good, with just two cases of mild side-effects. Clinical improvement was obtained in 19 (76%) patients. Parasitological cure, however, occurred only in a low percentage (25%). In some cases where initial success was observed, recrudescence occurred after some weeks or few months, but with retreatment again clinical improvement was obtained. Even if it does not lead to frequent parasite eradication, the good clinical results and tolerance permit us to consider paromomycin one of the few drugs effective for the treatment of cryptosporidial diarrhea in AIDS patients. Studies with maintainance therapy are indicated.